1. Technical Field
This invention relates to a continuous process for reducing the levels of certain nitrogen-containing compounds present in tobacco materials using microorganisms. Specifically, the present invention provides a process for reducing the levels of nitrates, nitrites and ammonium compounds via an aerobic assimilatory metabolic pathway employing conditions such that continuous, rather thar batch, operation is possible.
2. Description of Prior Art
It is generally recognized that smoking products having lowered amounts of oxides of nitrogen present in smoke are desirable. Therefore, a number of methods have been developed to reduce the delivery of oxides of nitrogen by smoking products. Among these techniques are various methods wherein the nitrate content of the tobacco is altered. For example, methods involving microbial treatment of tobacco to accomplish such nitrate reduction have been proposed.
Specifically in Gaisch et al. Belgian Pat. No. 886,445 published Aug. 14, 1978 and assigned to Fabriques de Tabac Reunies S.A. a process for degrading nitrates and nitrites in tobacco to nitrogen or ammonia compounds by means of microorganisms which would normally require oxygen, but are capable of anaerobic denitration is described. Gaisch et al. German Offenlegungsschrift No. 28 16427, filed Apr. 15, 1978 and published Nov. 9, 1978, describes a process for microbial degradation of nitrate, nitrite and other nitrogen containing compounds in tobacco. According to Gaisch et al., under nitrogen deficiency or oxygen deficiency conditions, the microorganisms employed obtain their nitrogen or oxygen requirements respectively from nitrate or nitrite degradation. The microorganisms which can be used in these two processes may be selected from the genus Aerobacter, Pseudomonas, Micrococcus or Escherichia, with Enterobacter aerogenes being specifically employed in the examples.
European Patent Application No. 79 300 706.3 published Oct. 31, 1979, describes a process for microbial reduction of nitrates in tobacco via a dissimilatory denitrification pathway whereby nitrogen gas is the end product. The microorganism specifically suggested for use in the process is Paracoccus denitrificans or Micrococcus denitrificans. Species of the genera Pseudomonas, Alcaligenes, Bacillus and Propionibacterium can also be employed.
Further U.S. Pat. No. 3,845,774 to Tso et al. describes tobacco treatment methods referred to as homogenized leaf curing wherein the tobacco is homogenized and incubated during curing in order to regulate the composition of the final product. Nitrate-nitrogen and total nitrogen are reduced somewhat; however, the amount of reduction is not as significant as that of the present process. Although Tso et al. allude to the fact that tobacco modification can be accomplished by the use of additional techniques during homogenization and incubation, such as enzyme and microbial action, no specific methods or means for reducing nitrate-nitrogen are suggested.
Gravely et al., U.S. Pat. No. 3,747,608 relates to a method for aerobic microbial digestion of pectin-bound plant material, specifically tobacco materials. Although the invention deals predominantly with methods for fibrillation tobacco materials using pectolytic enzyme-producing microorganisms, Examples 11 and 13 disclose data related to the concomitant denitration of tobacco using the mioroorganism Erwinia carotovora, ATCC 495. This microorganism is unsuitable for use in the present invention since pectolytic enzyme-producing microorganisms, such as Erwinia carotovora, destroy the structural integrity of the tobacco.
W. O. Atkinson et al. reported a reduction in various tobacco leaf components, including nitrate-nitrogen, by varying homogenization and incubation techniques during curing. (Abstract of Proceedings of the University of Kentucky Tobacco and Health Research Institute, Lexington, Ky., Conference Report 4, March 1973, pages 829-33.)
Denitration by means of microorganisms is also known outside the tobacco arts. Representative examples are U.S. Pat. No. 3,709,364 to Savage, U.S. Pat. No. 3,829,377 to Hashimoto, U.S. Pat. No. 4,039,438 to Anderson, and U.S. Pat. No. 4,043,936 to Francis et al. which describe denitrification of waste water using anaerobic bacteria to reduce the nitrate to nitrogen gas. Members of the Thiobacillus, Pseudomonas, Chromobacter, Bacillus and Clostridium genera are among the microorganisms which may be employed. In the Hashimoto patent the use of pressurized systems to increase the amount of methane available to the microorganisms and to facilitate liberation of the nitrogen gas by venting are suggested. The Anderson patent suggests conducting the process at ambient or atmospheric pressure. In the Francis patent the nitrogen gas passes through an exit out of the system. The Savage reference employs pressure to pass the effluent being treated through the filter containing the microorganisms.
Microorganisms have also been used to modify other tobacco components. For example, U.S. Pat. Nos. 4,037,609 and 4,038,993 to Geiss et al. disclose methods for reducing the nicotine content of tobacco by microbial treatment using microorganisms obtained from tobacco, including Pseudomonas putida and Cellulomonas sp. Aerobic fermentation techniques are employed wherein nicotine is degraded via microbial action to 3-succinoylpyridine. The latter microorganism is capable of reducing nitrate to nitrite and actively produces nitrogen gas. Similarly degradation of nicotine to 3-succinoylpyridine by means of the same microorganisms is described in U.S. Pat. No. 4,011,141 to Gravely et al. Lippmnan et al. U.S. Pat. No. 2,000,855 describes microbial denicotinization of tobacco by fermenting moist tobacco while adding acid to overcome the alkaline condition produced by fermentation. Alternatively the patent suggests removal of volatile bases by supplying an air current or employing suction. Fermentation was used to improve aroma and mellowness in U.S. Pat. No. 2,644,462 to Frankenburg and in U.S. Pat. No. 4,135,521 to Malan et al.
Further, U.S. Pat. No. 2,149,179 relates to an accelerated aging method for tobacco wherein the aging is effected by means of fermentation with exclusion of oxygen employing microorganisms capable of growing in the absence of oxygen. The microorganisms may be those which are bred on noble tobaccos or anaerobic yeasts. By means of the process, fermentation times of only days, rather than months are required. The purpose of the claimed fermentation process is to improve the bouquet of the tobacco. Nicotine content in the tobacco is also reduced. According to the patent, a prior process of Suchsland, which used microorganisms to decompose complex organic substances in tobacco into simpler compounds, did not prove practical since the oxidation effected by oxygen during the fermentation was ignored.
We have now unexpectedly discovered that by employing carefully controlled conditions, it is possible to effect denitration via an aerobic assimilatory metabolic pathway on a continuous basis. Specifically it has been discovered that by controlling the denitration conditions, it is possible to coordinate the microorganisms' growth rate with the tobacco extract treatment rate, whereby a denitration process is provided which is easily adapted to other continuous tobacco treatment processes, can be employed on a continuous basis for extended periods with relatively little or no supervision and permits treatment of greater amounts of tobacco extract and results in a higher production rate relative to batch processes. That is, the present process provides a method whereby nitrates, nitrites and ammonium compounds can be efficiently eliminated from tobacco via an assimilatory metabolic process on a large, technical scale under economical conditions, with a minimal requirement of manpower or energy and minimal addition to or transformations of the tobacco extract components, other than such denitration.